Pronase (EC 3.4.24.4)

Sweeney, Patricia J.; Walker, John M.

doi:10.1385/0-89603-234-5:271

Cited by 32 publications

(32 citation statements)

References 20 publications

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“…Total protein was extracted from OVCAR-8 cells treated with or without L-ASP (0.5 U/mL) and subsequently hydrolyzed using proteases to yield free amino acids (Sweeney and Walker 1993). Comparison of the asparagine levels between the two groups showed no differences between vehicle- and L-ASP-treated cells (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

et al. 2014

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L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP’s anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC–MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells. When we examined the response of OVCAR-8 ovarian cancer cells in culture to L-ASP, asparagine was depleted from the medium within seconds. Interestingly, intracellular asparagine was also depleted rapidly, and the mechanism was suggested to involve rapid export of intracellular asparagine followed by rapid conversion to aspartic acid by L-ASP. We also found that L-ASP-induced cell death was more closely associated with glutamine concentration than with asparagine concentration. Time-course analysis revealed the dynamics of amino acid metabolism after feeding cells with fresh medium. Overall, this study provides new insight into L-ASP’s mechanism of action, and the optimized analytical method can be extended, with only slight modification, to other metabolically active amino acids, related compounds, and a range of cultured cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-014-0634-1) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

et al. 2014

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“…It is also apparent that the propensity for hydrolysis of this type of amide bond is unaffected by the configuration of the b 3 hAla side-chain group. Analogy can be drawn to the fact that pronase is unusual in its ability to cleave a-peptidic bonds of damino acids [36]. The pH of the assay solutions (pH 7.8 and 9.0) did not influence the overall outcome of the reaction, although endopeptidase components of pronase are more prolific at lower pH values and exopeptidases more active under basic conditions.…”

Section: B-peptidesmentioning

confidence: 93%

“…Pronase (EC 3.4.24.4) consists of a group of at least ten proteolytic enzymes obtained from the culture supernatant of Streptomyces griseus K-1. Being a mixture of endo-and exopeptidases, pronase has broad (in reality low) specificity and cleaves nearly all peptide bonds; it is known to contain five serine-type proteases, two Zn 2 endopeptidases, two Zn 2 leucine aminopeptidases, and one Zn 2 carboxypeptidase [36]. As such, there is no single pH optimum; therefore, assays involving pronase were performed at pH 7.8 and 9.0 in this study.…”

Section: Choice Of Enzymesmentioning

confidence: 99%

The Proteolytic Stability of ‘Designed’β-Peptides Containingα-Peptide-Bond Mimics and of Mixedα,β-Peptides: Application to the Construction of MHC-Binding Peptides

Hook

Bindschädler

Mahajan

et al. 2005

C&B

117

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Dedicated to Professor Piero Salvadori, University of Pisa, on the occasion of his 70th birthday Whereas a-peptides are rapidly degraded in vivo and in vitro by a multitude of peptidases, substrates constructed entirely of or incorporating homologated a-amino acid (i.e., b-amino acid) units exhibit a superior stability profile. Efforts made so far to proteolytically hydrolyze a bÀb peptide bond have not proved fruitful; a study aimed at breaching this proteolytic stability is discussed here. A series of such bonds have been designed with side-chain groups similar in relative positions (constitution) and three-dimensional arrangements (configuration) as found about a-peptidic amide bonds. Increasing the prospect for degradation would permit the tuning of b-peptide stability; here, however, no cleavage was observed (1, 2, 4 ± 6, Table 1). Peptides comprised of a-and b-amino acids (mixed a,b-peptides, 8 ± 11) are expected to benefit from both recognition by a natural receptor and a high level of proteolytic stability, ideal characteristics of pharmacologically active compounds. b 3 -Peptides containing a-amino acid moieties at the N-terminus are degraded, albeit slowly, by several peptidases. Of particular interest is the ability of pronase to cleave an aÀb peptide bond, namely that of aAlaÀb 3 hAla. Significantly, successful hydrolysis is independent of the configuration of the b-amino acid. Some of the a,b-peptides discussed here are being investigated for their binding affinities to class I MHC proteins. The computer-programming steps required to prepare a,b-peptides on an automated peptide synthesizer are presented.

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“…either phosphorylated or phosphonylated) by numerous organophosphorus compounds (OP) [19][20][21][22][23][24] . After enzymatic degradation using pronase [18] , small biomarkers (DPAET-Cys-Pro, DPAET-CP and Pro-Cys-DPAET, PC-DPAET) were detected by microbore liquid chromatographic separation and electrospray ionization with subsequent high-resolution tandem mass spectrometric detection (μLC-ESI MS/HR MS). After enzymatic degradation using pronase [18] , small biomarkers (DPAET-Cys-Pro, DPAET-CP and Pro-Cys-DPAET, PC-DPAET) were detected by microbore liquid chromatographic separation and electrospray ionization with subsequent high-resolution tandem mass spectrometric detection (μLC-ESI MS/HR MS).…”

Section: Introductionmentioning

confidence: 99%

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin

Kranawetvogl

Küppers

Gütschow

et al. 2017

Drug Testing and Analysis

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Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd.

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Pronase (EC 3.4.24.4)

Cited by 32 publications

References 20 publications

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

The Proteolytic Stability of ‘Designed’β-Peptides Containingα-Peptide-Bond Mimics and of Mixedα,β-Peptides: Application to the Construction of MHC-Binding Peptides

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin

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